Vangipuram S. Rangan

Cloning, Expression, and Characterization of a Human 4′-Phosphopantetheinyl Transferase with Broad Substrate Specificity

A single candidate 4′-phosphopantetheine transferase, identified by BLAST searches of the human genome sequence data base, has been cloned, expressed, and characterized. The human enzyme, which is expressed mainly in the cytosolic compartment in a wide range of tissues, is a 329-residue, monomeric protein. The enzyme is capable of transferring the 4′-phosphopantetheine moiety of coenzyme A to a conserved serine residue in both the acyl carrier protein domain of the human cytosolic multifunctional fatty acid synthase and the acyl carrier protein associated independently with human mitochondria. The human 4′-phosphopantetheine transferase is also capable of phosphopantetheinylation of peptidyl carrier and acyl carrier proteins from prokaryotes. The same human protein also has recently been implicated in phosphopantetheinylation of the α-aminoadipate semialdehyde dehydrogenase involved in lysine catabolism (Praphanphoj, V., Sacksteder, K. A., Gould, S. J., Thomas, G. H., and Geraghty, M. T. (2001) Mol. Genet. Metab. 72, 336–342). Thus, in contrast to yeast, which utilizes separate 4′-phosphopantetheine transferases to service each of three different carrier protein substrates, humans appear to utilize a single, broad specificity enzyme for all posttranslational 4′-phosphopantetheinylation reactions.

The Human Thioesterase II Protein Binds to a Site on HIV-1 Nef Critical for CD4 Down-regulation

A HIV-1 Nef affinity column was used to purify a 35-kDa Nef-interacting protein from T-cell lysates. The 35-kDa protein was identified by peptide microsequence analysis as the human thioesterase II (hTE) enzyme, an enzyme previously identified in a yeast two-hybrid screen as a potential Nef-interacting protein. Immunofluorescence studies showed that hTE localizes to peroxisomes and that coexpression of Nef and hTE leads to relocalization of Nef to peroxisomes. Interaction of Nef and hTE was abolished by point mutations in Nef at residues Asp108, Leu112, Phe121, Pro122, and Asp123. All of these mutations also abrogated the ability of Nef to down-regulate CD4 from the surface of HIV-infected cells. Based on the x-ray and NMR structures of Nef, these residues define a surface on Nef critical for CD4 down-regulation. A subset of these mutations also affected the ability of Nef to down-regulate major histocompatibility complex class I. These results, taken together with previous studies, identify a region on Nef critical for most of its known functions. However, not all Nef alleles bind to hTE with high affinity, so the role of hTE during HIV infection remains uncertain.

Dibromopropanone Cross-linking of the Phosphopantetheine and Active-site Cysteine Thiols of the Animal Fatty Acid Synthase Can Occur Both Inter- and Intrasubunit REEVALUATION OF THE SIDE-BY-SIDE, ANTIPARALLEL SUBUNIT MODEL

The objective of this study was to test a new model for the homodimeric animal FAS which implies that the condensation reaction can be catalyzed by the amino-terminal β-ketoacyl synthase domain in cooperation with the penultimate carboxyl-terminal acyl carrier protein domain of either subunit. Treatment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; none of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the β-ketoacyl synthase domain (C161A) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151A). A double affinity-labeling strategy was used to isolate dimers that carried one or both mutations on one or both subunits; the heterodimers were treated with dibromopropanone and analyzed by a combination of sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtration, and matrix-assisted laser desorption mass spectrometry. Thus the two slowest moving of these species, which accounted for 45 and 15% of the total, were identified as doubly and singly cross-linked dimers, respectively, whereas the fastest moving species, which accounted for 35% of the total, was identified as originating from internally cross-linked subunits. These results show that the two polypeptides of the fatty acid synthase are oriented such that head-to-tail contacts are formed both between and within subunits, and provide the first structural evidence in support of the new model.

Engineering of an Active Animal Fatty Acid Synthase Dimer with Only One Competent Subunit

Animal fatty acid synthases are large polypeptides containing seven functional domains that are active only in the dimeric form. Inactivity of the monomeric form has long been attributed to the obligatory participation of domains from both subunits in catalysis of substrate loading and condensation reactions. However, we have engineered a fatty acid synthase containing one wild-type subunit and one subunit compromised by mutations in all seven functional domains that is active in fatty acid synthesis. This finding indicates that a single subunit, in the context of a dimer, is able to catalyze the entire biosynthetic pathway and suggests that, in the natural complex, each of the two subunits forms a scaffold that optimizes the conformation of the companion subunit.

Expression in Escherichia coli and Refolding of the Malonyl-/Acetyltransferase Domain of the Multifunctional Animal Fatty Acid Synthase

A cDNA encoding residues 429-815 of the multifunctional rat fatty acid synthase has been expressed in Escherichia coli and the recombinant protein refolded in vitro as a catalytically active malonyl-/acetyltransferase. Kinetic properties of the refolded recombinant enzyme were indistinguishable from those of a transferase preparation derived from the natural fatty acid synthase by limited proteolysis, indicating that the transferase domain is capable of folding correctly as an independent protein. Replacement of the active site Ser-581 (full-length fatty acid synthase numbering) with alanine completely eliminated catalytic activity, whereas replacement with cysteine resulted in retention of about 1% activity. The wild type transferase was extremely susceptible to inhibition by diethyl pyrocarbonate, and protection against inhibition was afforded by both malonyl- and acetyl-CoA. Replacement of the highly conserved residue His-683 with Ala reduced activity by 99.95%, and the residual activity was relatively unaffected by diethyl pyrocarbonate. The rate of acylation of the active site serine residue was also reduced by several orders of magnitude in the His-683 → Ala mutant. These results indicate that His-683 plays an essential role in catalysis, likely by accepting a proton from the active site serine, thus increasing its nucleophilicity.

Chapter 6 Fatty acid synthesis in eukaryotes

Publisher Summary This chapter focuses primarily on the structure of fatty acids, their mechanism of action, and regulation of the enzymes responsible for the biosynthesis of long-chain saturated fatty acids, de novo. In eukaryotes, the enzymes required for fatty acid synthesis de novo are integrated into large multifunctional polypeptides that are located in the cytosol. However, the domain organization and overall molecular architecture of the animal and yeast complexes are quite different. Fatty acids fulfill several crucial roles in animals, represent a major storage form of energy, and provide an essential structural component of membranes. In animals, the catalytic components required for the entire fatty acid biosynthetic pathway are integrated into two multifunctional polypeptides: acetyl- coenzyme A (CoA) carboxylase (ACC) and fatty acid synthase (FAS). The expression of both enzymes is regulated at the transcriptional level, in a tissue-specific manner, and in response to various developmental, nutritional, and hormonal signals. Substantial progress has been made to identify the various trans-acting factors that regulate transcription of the ACC and FAS genes in response to nutritional stimuli, and many of the remaining details of these processes are likely to be elaborated in the near future.